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Pressure-Induced Protein Unfolding in the Ternary System AOT-Octane-Water Is Different from that in Bulk Water Filip Meersman,†,* Carolien Dirix,§ Stepan Shipovskov,¶ Natalia L. Klyachko,| and Karel Heremans§,* Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium, Department of Molecular Biophysics, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden, and Department of Chemical Enzymology, Faculty of Chemistry, Moscow State University, 119899 Moscow, Russia Received December 1, 2004. In Final Form: January 31, 2005 In a cellular environment, the presence of macromolecular cosolutes and membrane interfaces can influence the folding-unfolding behavior of proteins. Here we report on the pressure stability of R-chymotrypsin in the ternary system bis(2-ethylhexyl)sodium sulfosuccinate-octane-water using FTIR spectroscopy. The ternary system forms anionic reverse micelles which mimic cellular conditions. We find that inclusion of a single protein molecule in a reverse micelle does not alter its conformation. When pressurized in bulk water, R-chymotrypsin unfolds at 750 MPa into a partially unfolded structure. In contrast, in the ternary system, the same pressure increase induces a random coil-like unfolded state, which collapses into an amorphous aggregate during the decompression phase. It is suggested that the unfolding pathway is different in a cell-mimicking environment due to the combined effect of multiple factors, including confinement. A phase transition of the reverse micellar to the lamellar phase is thought to be essential to provide the conditions required for unfolding and aggregation, though the unfolding is not a direct result of the phase transition. Our observations therefore suggest that membranes may cause the formation of alternative conformations that are more susceptible to aggregation.
Introduction Most biochemical reactions, such as protein folding and unfolding, are studied in vitro in dilute aqueous solutions. However, in vivo these reactions occur in a highly crowded environment. Macromolecular crowding will affect the equilibria and rates of these reactions.1-3 In addition, there is the presence of lipid membranes which interact with many proteins, whether these are membrane-bound or not.4,5 It has been demonstrated that membranes can influence the folding-unfolding reactions of proteins and change the protein conformation compared to the solution state.6,7 Of particular interest is the possible role of lipid membranes in a number of human diseases such as Creutzfeldt-Jakob disease, light chain amyloidosis, and Alzheimer’s disease.8-11 These diseases are characterized * Authors to whom correspondence should be addressed. Tel: 0032 16 32 71 59 (K.H.); 0044 1223 76 38 45 (F.M.). Fax: 0032 16 32 79 82 (K.H.); 0044 1223 76 38 49 (F.M.). E-mail:
[email protected] (K.H.);
[email protected] (F.M.). † University of Cambridge. § Katholieke Universiteit Leuven. ¶ Lund University. | Moscow State University. (1) van den Berg, B.; Ellis, R. J.; Dobson, C. M. EMBO J. 1999, 18, 6927. (2) Minton, A. P. Curr. Opin. Struct. Biol. 2000, 10, 34. (3) Ellis, R. J. Trends Biochem. Sci. 2001, 26, 597. (4) Epand, R. M. Biochim. Biophys. Acta 1998, 1376, 353. (5) Klyachko, N. L.; Levashov, P. A.; Ko¨hling, R.; Woenckhaus, J.; Balny, C.; Winter, R.; Levashov, A. V. In Trends in High-Pressure Bioscience and Biotechnology; Hayashi, R., Ed.; Elsevier Science: Amsterdam, 2002; p 159. (6) Sanghera, N.; Pinheiro, T. J. T. Protein Sci. 2000, 9, 1194. (7) Sharp, J. S.; Forrest, J. A.; Jones, R. A. L. Biochemistry 2002, 41, 15810. (8) Terzi, E.; Ho¨lzemann, G.; Seelig, J. J. Mol. Biol. 1995, 252, 633. (9) Zhu, M.; Souillac, P. O.; Ionescu-Zanetti, C.; Carter, S. A.; Fink, A. L. J. Biol. Chem. 2002, 277, 50914. (10) Sanghera, N.; Pinheiro, T. J. T. J. Mol. Biol. 2002, 315, 1241.
by the deposition of threadlike, ordered proteinaceous aggregates called amyloid fibrils. It has been demonstrated for several disease-related proteins that their assembly into fibrillar structures can be catalyzed by anionic membranes.9,12,13 In this work, we explore the pressure-induced unfolding of R-chymotrypsin in the ternary system bis(2-ethylhexyl)sodium sulfosuccinate (AOT)-octane-water. This system spontaneously forms reverse micelles, which are waterin-oil droplets consisting of spheroidal assemblies of AOT whereby the polar headgroup of the AOT molecule is directed toward the water phase in the interior of the sphere and the hydrocarbon tails are in contact with the bulk organic solvent. The AOT layer can be considered a membrane mimic, thereby providing a water-membrane interface.14-16 Moreover, this ternary system has two additional advantages compared to other membrane mimics such as sodium dodecyl sulfate micelles and lipid vesicles, which are oil-in-water systems. Because the size of reverse micelles depends solely on the molar ratio of water to surfactant (wo), it is possible to create conditions in which a single protein molecule surrounded by a water shell is incorporated in a reverse micelle. As such, reverse micelles represent (i) a confined geometry similar to that induced by intracellular crowding,17 as well as (ii) a low (11) Ehehalt, R.; Keller, P.; Haass, C.; Thiele, C.; Simons, K. J. Cell Biol. 2003, 160, 113. (12) Chirita, C. N.; Necula, M.; Kuret, J. J. Biol. Chem. 2003, 278, 25644. (13) Munishkina, L. A.; Phelan, C.; Uversky, V. N.; Fink, A. L. Biochemistry 2003, 42, 2720. (14) Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 947, 209. (15) Martinek, K.; Klyachko, N. L.; Kabanov, A. V.; Khmenlnitsky, Yu. L.; Levashov, A. V. Biochim. Biophys. Acta 1989, 981, 161. (16) Nicot, C.; Waks, M. Biotechnol. Genet. Eng. Rev. 1995, 13, 267. (17) Shastry, M. C. R.; Eftink, M. R. Biochemistry 1996, 35, 4094.
10.1021/la0470481 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/02/2005
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water system.18 The latter is also a cellular characteristic, as it is known that, although up to 70-80% of the cell consists of water, many macromolecules in the cell are surrounded by only a small number of hydration layers.19 It should be emphasized that different lipid phases, such as the reverse micelle and the lamellar and hexagonal phases, occur in biological membranes, where they play a role in regulating enzyme activity.4,20 Apart from their cell-like features reverse micelles are also optically transparent, allowing spectroscopic studies. R-Chymotrypsin is a 25 kDa serine protease consisting of three chains connected by five disulfide bonds.21 Its catalytic activity in the AOT-octane-water system has been extensively characterized.20,22,23 This has also included a study of the activity as a function of pressure.24,25 However, to our knowledge, this is the first study of the conformational behavior of R-chymotrypsin in reverse micelles exposed to high pressure (up to 1.0 GPa). Our work demonstrates that under conditions mimicking a physiological environment pressure-induced unfolding of R-chymotrypsin results in an unfolded state that lacks any ordered secondary structure. In contrast, in bulk water, R-chymotrypsin undergoes only partial unfolding, with a significant amount of secondary structure remaining, indicating that the unfolding process is different under these conditions. Moreover, an amorphous aggregate is formed during the decompression phase in the ternary system, which is not the case in bulk water. Such an effect is not commonly observed after pressure treatment and is likely associated with the presence of the membrane mimic. This provides further evidence for the possible role of membranes in the development of disease-associated aggregates. In order for the unfolding and aggregation to be able to take place, the ternary system has to undergo a phase transition to a lamellar phase. The mechanistic aspects of unfolding and aggregation will be discussed in detail. Experimental Section Sample Preparation. n-Octane and AOT were purchased from Sigma (Belgium), DCl was obtained from Aldrich (Belgium), and D2O was from Cambridge Isotope Laboratories, Inc. (Andover, MA). Bovine pancreatic R-chymotrypsin was purchased from Sigma (Belgium) and was used without further purification. The protein was dissolved in D2O. The pD of the protein solution was 3.0 (corrected for deuterium isotope effect). For the experiment at pD 2.1, DCl was added to the solution until the desired pD was reached. The samples were stored overnight to ensure complete H/D-exchange of all solvent-accessible protons. The system AOT-octane-water was obtained by dissolving AOT in octane (1.0 M AOT) followed by addition of a 20% w/v enzyme solution and/or D2O to yield the desired molar ratio of water to surfactant (AOT). The R-chymotrypsin concentration in pure D2O was 5% w/v. (18) Tuena de Go´mez-Puyou, M.; Go´mez-Puyou, A. Crit. Rev. Biochem. Mol. Biol. 1998, 33, 53. (19) Mentre´, P. Cell. Mol. Biol. 2001, 47, 709. (20) Klyachko, N. L.; Levashov, A. V.; Kabanov, A. V.; Khmelnitsky, Yu. L.; Martinek, K. In Kinetics and catalysis in microheterogeneous systems; Gratzel M., Kalyanasundaram, K., Eds.; Marcel Dekker: New York, 1991; p 135. (21) Birktoft, J. J.; Blow, D. M.; Henderson, R.; Steitz, T. A. Philos. Trans. R. Soc. London B 1970, 257, 67. (22) Bru, R.; Sancher-Ferrer, A.; Garcia-Carmona, F. Biochem. J. 1995, 310, 721. (23) Dorovska-Taran, V.; Veeger, C.; Visser, A. J. Eur. J. Biochem. 1993, 218, 1013. (24) Mozhaev, V. V.; Bec, N.; Balny, C. Biochem. Mol. Biol. Int. 1994, 34, 191. (25) Rariy, R. V.; Bec, N.; Saldana, J.-L.; Nametkin, S. N.; Mozhaev, V. V.; Klyachko, N. L.; Levashov, A. V.; Balny, C. FEBS Lett. 1995, 364, 98.
Meersman et al. Fourier Transform Infrared Spectroscopy. High hydrostatic pressure was generated in a diamond anvil cell (www.diacell.co.uk). Barium sulfate was used as an internal pressure standard in all cases.26 The pressure cell was connected to a thermostat to keep the temperature constant at 25 °C. The infrared spectra were recorded on a Bruker IFS66 FTIR spectrometer equipped with a liquid-nitrogen-cooled broad-band mercury-cadmium-telluride solid-state detector. Typically, 256 interferograms were co-added after registration at a resolution of 2 cm-1. The AOT-octane contribution to the spectrum was subtracted, and the resulting spectra were smoothed and baseline corrected. Secondary structure analysis of the native states was done by Gaussian curve-fitting of the resolution-enhanced spectra (using GRAMS/AI.7 from Thermo Galactic). The wavenumbers of the band centers found in the second-derivative spectra were used as the starting parameters. Resolution enhancement was achieved by Fourier self-deconvolution, a mathematical technique of band narrowing. A band-narrowing parameter (γ) of 10 and a Bessel smoothing function (70%) were used (unless mentioned otherwise).
Results Under the conditions used in this study, the ternary system AOT-octane-water spontaneously forms reverse micelles (at 25 °C and 0.1 MPa). The stability of R-chymotrypsin was investigated in the presence of variable amounts of water, with wo ranging from 5 to 40. As the results did not show a dependence on wo, we limit the following description to the experiments at wo ) 10. A molar ratio of water-to-surfactant of 10 is most often used in studies of R-chymotrypsin because (i) this results in the entrapment of a single protein molecule in each reverse micelle and (ii) the enzyme reaches its activity optimum close to this wo value (at pH ≈ 8).20 Note that, in the present work, at pD 3, R-chymotrypsin is not enzymatically active but remains conformationally intact. Incorporation of r-Chymotrypsin in an AOTWater-Octane System at 25 °C and 0.1 MPa Does Not Alter Its Conformation. To compare the stability of R-chymotrypsin in water and in reverse micelles, it is important to analyze its native structure under both conditions first. It is known that entrapment of proteins in reverse micelles can induce conformational changes of variable magnitude depending on factors such as the nature of the protein, the type of surfactant, and wo value.16 Such a conformational change might affect the pressuretemperature stability. The deconvoluted amide I′ bands of R-chymotrypsin in water and in reverse micelles at 25 °C and 0.1 MPa are shown in Figure 1. It can be seen that the spectra are very similar. The secondary structure content has been determined by curve fitting. Nine bands are found to contribute to the amide I′ area. The bands at 1621, 1629, 1637 and 1673 cm-1 can be assigned to β-sheet structure; those around 1647, 1655 and 1664-1682-1690 cm-1 are attributed to disordered, R-helix, and turn structures, respectively.27-29 Bands below 1610 cm-1 are due to sidechain vibrations. The relative contributions of each type of structure are summarized in Table 1. The band positions and their relative contributions are in good agreement with earlier work on R-chymotrypsin in solution.27,29-31 (26) Wong, P. T. T.; Moffat, D. J. Appl. Spectrosc. 1989, 43, 1279. (27) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469. (28) Ismail, A. A.; Mantsch, H. H.; Wong, P. T. T. Biochim. Biophys. Acta 1992, 1121, 183. (29) Qinglong, C.; Huizhou, L.; Jiayong, C. Biochim. Biophys. Acta 1994, 1206, 247. (30) Dong, A.; Huang P.; Caughey, W. S. Biochemistry 1990, 29, 3303. (31) Carrasquillo, K. G.; Sanchez, C.; Griebenow, K. Biotechnol. Appl. Biochem. 2000, 31, 41.
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Figure 1. The amide I′ band of R-chymotrypsin in water (A) and in the ternary system AOT-water-octane (B). Dashed lines are the individual Gaussian bands fitted to the spectra. The spectra were Fourier self-deconvoluted before curve-fitting. Table 1. Secondary Structure Content of r-Chymotrypsin in Water and in the Ternary System AOT-Water-Octane (wo ) 10) at 25 °C and 0.1 MPa secondary structure (%) D2O RMa a
R-helix
β-sheet
disordered
turn
11 ( 1 13 ( 2
49 ( 4 49 ( 5
18 ( 2 15 ( 2
21 ( 1 23 ( 3
RM: reverse micelle.
Our data therefore suggest that the secondary structure of R-chymotrypsin is not significantly perturbed by entrapment in reverse micelles at ambient temperature and pressure. Furthermore, under both conditions, a significant band around 1550 cm-1 is still present (data not shown). This is the so-called amide II band which is mainly due to the backbone N-H bending vibration and is very sensitive to H/D exchange. The exchange causes a shift of the amide II band from 1550 to 1450 cm-1.32 Its presence indicates that in neither case all the hydrogen atoms have been exchanged, suggesting that even the tertiary structure of R-chymotrypsin is, if at all, not strongly affected by the inclusion in a reverse micelle. Pressure Induces a Larger Degree of Unfolding in the Ternary System AOT-Water-Octane Than in Bulk Water (at 25 °C). Figure 2A shows the pressureinduced changes in the amide I′ band of R-chymotrypsin in the ternary system. It can be seen that the intense band around 1637 cm-1 (β-sheet) gradually disappears. The transition midpoint can be found at approximately 750 MPa. At high pressure (∼1.0 GPa), a broad, feature(32) Haris, P. I.; Chapman, D. Biopolymers 1995, 37, 251.
Figure 2. Stacked plot of the pressure-induced changes in the amide I′ band of R-chymotrypsin in the ternary system: (A) pressure increase, (B) pressure decrease. The top spectrum is obtained after ∼12 h at 0.1 MPa. Pressures are indicated in MPa. The difference spectrum of the amide I′ band of R-chymotrypsin at 990 and 0.1 MPa is shown in (C). Spectra were baseline-corrected and area-normalized prior to subtraction.
less, and symmetric band centered around 1646 cm-1 can be observed. A difference spectrum of the native state and pressure-induced states clearly indicates the loss of native β-sheet and the appearance of disordered structure, as well as bands around 1658 and 1666 cm-1 (Figure 2C). The latter indicate the formation of irregular loop and turn structures, respectively. These features are typical of a highly unfolded protein. A similar band shape can be observed for a natively unfolded protein such as R-synuclein.13 The pressure effect on the protein in bulk water is shown in Figure 3. In this case, the spectrum of the unfolded state at ∼1.2 GPa is less symmetric and its maximum of intensity can be found near 1640 cm-1. This can be seen in Figure 4, which also shows the spectrum of the unfolded state in the ternary system for comparison. The band shape in bulk water is almost identical to that found for
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Figure 4. Pressure-unfolded states of R-chymotrypsin in water (top) and in the ternary system (bottom). Deconvoluted spectra are shown. The spectra were normalized for the same overall intensity.
Figure 3. Stacked plot of the pressure-induced changes in the amide I′ band of R-chymotrypsin in bulk water: (A) pressure increase, (B) pressure decrease. Pressures are indicated in MPa. The difference spectrum of the amide I′ band of R-chymotrypsin at 1499 and 0.1 MPa is shown in (C). Spectra were baselinecorrected and area-normalized prior to subtraction.
chymotrypsinogen in Tris buffer at ∼1.3 GPa.33 A number of distinct components can still be observed after deconvolution of the spectrum, indicating that the unfolded state retains some ordered secondary structure (not shown). However, part of the β-sheet in the unfolded state is nonnative in character because there is the clear disappearance of the 1637 cm-1 band, as well as an increased absorbance of the band area below 1630 cm-1 (Figure 3C). The formation of the latter is in agreement with the effect of pressure on chymotrypsinogen28 and has also been observed in the case of myoglobin.34 The spectra therefore suggest that the unfolded states in water and in the ternary system are different. Note, however, that the pressure stability of the protein is approximately the same under both conditions (p1/2 ≈ 750 MPa). The same result was obtained at pD 2.1. (33) Wong, P. T. T. Can. J. Chem. 1991, 69, 1699. (34) Meersman, F.; Smeller, L.; Heremans, K. Biophys. J. 2002, 82, 2635.
Upon decompression, it can be observed that in aqueous solution the amide I′ band maximum shifts to 1637 cm-1, indicative of the formation of nativelike β-sheet structure (Figure 3B). However, the spectrum does not completely assume the shape corresponding to the native state. This suggests that the unfolding induced by pressure is only partially reversible. Indeed, curve-fitting indicates that this state contains more irregular structure (63 ( 5%) and less β-sheet structure (37 ( 5%) than the native state. In contrast, in the ternary system R-chymotrypsin adopts a new conformation that is different from the native and unfolded states. As can be seen from Figure 2B, the amide I′ band is dominated by a well-resolved band at 1629 cm-1 which is accompanied by a weak absorption around 1687 cm-1. The appearance of these two new bands can be interpreted in terms of the formation of amorphous aggregates rich in β-sheet structure. Generally, in bulk solvent, these bands appear at ∼1616 and 1683 cm-1, respectively,28,34 indicating that in the present case the bands have undergone a blue-shift. This is likely due to a change in relative permittivity, which would arise from a close association of the protein with the membrane mimic. Such an effect has been observed before, for instance, for the adsorption of β-lactoglobulin to dimyristoylphosphatidylglycerol bilayers.35 Note that the aggregation bands do not appear until the pressure is released to approximately 200 MPa. High hydrostatic pressure is known to dissociate protein aggregates and will oppose the formation of aggregates at high pressure. Dissociation of native oligomers or aggregates generally occurs at a pressure of 200-300 MPa. This phenomenon is assumed to be due to the fact that the hydrophobic and electrostatic interactions are weakened at high pressure. Aggregation in the decompression phase is generally found to occur at pressures below this threshold.36 Discussion It is important to emphasize that at 25 °C and 0.1 MPa the R-chymotrypsin molecule is located in the water pool37 and does not adsorb to AOT. The latter would most likely induce a conformational rearrangement of the protein, as observed in many cases.6,8,38 Such a rearrangement has (35) Lefe`vre, T.; Subirade, M. Biochim. Biophys. Acta 2001, 1549, 37. (36) Meersman, F.; Heremans, K. Biophys. Chem. 2003, 104, 297. (37) Battistel, E.; Luisi, P. L.; Rialdi, G. J. Phys. Chem. 1988, 92, 6680. (38) Giacomelli, C. E.; Norde, W. Biomacromolecules 2003, 4, 1719.
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been suggested to play a role in, for instance, the insertion of proteins in membranes and the membrane facilitated aggregation of proteins. However, we find no evidence of such a structural perturbation upon insertion of R-chymotrypsin in reverse micelles, which is in accordance with previous work (reviewed by Nicot and Waks16). Note that the work of Qinglong et al.29 is often cited to point out the occurrence of significant structural changes. However, this paper shows that the authors found little structural changes in absolute terms, although the relative changes (in %) were rather large. Influence of a Cell-Mimicking Environment on the Pressure-Induced Unfolding Process. High hydrostatic pressure is well established as a physical perturbation factor capable of unfolding proteins.39 Here we have shown that in bulk water the pressure-unfolded state of R-chymotrypsin retains a significant amount of secondary structure, whereas under conditions resembling more closely those within the cell, the protein adopts a random coil-like conformation, albeit constrained by the presence of the disulfide bonds. One may assume that the different outcome in the presence of an AOT-octane interface reflects that the protein undergoes another unfolding mechanism. Indeed, the intermediate-like state obtained at high pressure in bulk water cannot be observed during the pressure unfolding of R-chymotrypsin in the ternary system. This rules out that the pathway in the ternary system is a mere extension of that in bulk water. In the following, we will argue that in the presence of the membrane mimic AOT a number of other factors can influence the pressure unfolding of the protein. First, the negatively charged headgroups of AOT attract protons from solution, thereby lowering the pH near the interface.6 The decrease is expected not to exceed 2 pH units in salt-free solutions,13 and this effect is probably smaller in an acidic solution. To assess the importance of this process, a possible pH decrease near the interface was simulated by performing a control experiment at pD 2.1 in bulk water, which is 1 pH unit lower than in the original experiment. This did not reveal any differences from the experiment at pD 3.0, suggesting that the pH effect alone plays only a minor role. Second, the aqueous pool of reverse micelles is assumed to contain different water populations, the properties of which can be of importance in determining the strength of the hydrophobic effect and of the hydrogen bonds.17 In fact, one particular property of water is the relative permittivity, , which is lower near the interface than in bulk water.13,16 High pressure will not affect this destabilizing factor as the effect of pressure on the relative permittivity of water is negligible ((δ ln )/(δp) ≈ 0.5 10-7 MPa-1).40 A low permittivity can enhance the strength of the hydrogen bonds and the electrostatic interactions. The latter can also form between the protein and the anionic membrane mimic since the protein is positively charged at pH 3 (pI of R-chymotrypsin ≈ 8.5). In general, the electrostatic interactions have a destabilizing effect on the protein.17 However, pressure disrupts electrostatic interactions due to the electrostriction effect.41 Thus, their contribution to the unfolding mechanism is expected to be small. An attempt was made to probe the effect of the electrostatic interactions by screening the charges, but the addition of 100 mM NaCl resulted in the salting out of the protein prior to incorporation in reverse micelles. Third, as pointed (39) Balny, C.; Masson, P.; Heremans, K. Biochim. Biophys. Acta 2002, 1595, 3. (40) Srinivasan, K. R.; Kay, R. L. J. Chem. Phys. 1974, 60, 3645. (41) Boonyaratanakornkit, B. B.; Beum Park, C.; Clark, D. S. Biochim. Biophys. Acta 2002, 1595, 235.
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out previously by Shastry and Eftink,17 a protein-octane interaction cannot be excluded. This could occur as a result of the dynamics of the system.18,25 It can be argued, though, that this interaction can only be a secondary driving force of the unfolding, as it is known that suspensions of lyophilized R-chymotrypsin in octane are still enzymatically active.42 This implies that the conformation of the protein cannot be significantly perturbed. Nevertheless, the apolar phase might promote a greater extent of unfolding by accommodating the hydrophobic regions of the protein at its interface with water, thereby minimizing the unfavorable interaction of hydrophobic groups with water. On the other hand, it can also be argued that at the pressures where the unfolding takes place (>200 MPa) the ternary system is in a lamellar phase (see below), which is characterized by a denser packing of the AOT molecules.43 As a consequence, the system will be less dynamic, creating fewer opportunities for protein-octane interactions. A more likely, alternative hydrophobic interaction would occur if the protein (or parts thereof) would insert itself in between the AOT molecules. As we will discuss below, changes in the system geometry could contribute to this phenomenon. A final factor that could possibly influence the degree of unfolding is the confined geometry experienced by the protein. Confinement shifts the equilibrium toward the state occupying the smallest volume, favoring a nativelike structure over a more unfolded conformation.2,3 This is generally true in cases where the space boundaries are set by an invariable system such as pores in silica glasses. However, if, under pressure, the geometry of the system changes in such a way that the unfolded state becomes the only conformation that can be optimally accommodated in the available microenvironment, then confinement may contribute to the unfolding. This is supported by a theoretical analysis of the effects of confinement and crowding on protein stability by Zhou.44,45 Whereas confinement can induce a significant protein stabilization (15 kcal/mol or more), crowding seems to have a much smaller effect (
